ROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPARα -mediated inhibition of...

Drukala et al. Molecular Cancer 2010, 9:159http://www.molecular-cancer.com/content/9/1/159

Open AccessR E S E A R C H

ResearchROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPARα -mediated inhibition of Glioma cell motility in vitroJustyna Drukala3, Katarzyna Urbanska2, Anna Wilk1, Maja Grabacka4, Ewa Wybieralska3, Luis Del Valle1, Zbigniew Madeja3 and Krzysztof Reiss*1

AbstractBackground: Glioblastomas are characterized by rapid cell growth, aggressive CNS infiltration, and are resistant to all known anticancer regimens. Recent studies indicate that fibrates and statins possess anticancer potential. Fenofibrate is a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that can switch energy metabolism from glycolysis to fatty acid β-oxidation, and has low systemic toxicity. Fenofibrate also attenuates IGF-I-mediated cellular responses, which could be relevant in the process of glioblastoma cell dispersal.

Methods: The effects of fenofibrate on Glioma cell motility, IGF-I receptor (IGF-IR) signaling, PPARα activity, reactive oxygen species (ROS) metabolism, mitochondrial potential, and ATP production were analyzed in human glioma cell lines.

Results: Fenofibrate treatment attenuated IGF-I signaling responses and repressed cell motility of LN-229 and T98G Glioma cell lines. In the absence of fenofibrate, specific inhibition of the IGF-IR had only modest effects on Glioma cell motility. Further experiments revealed that PPARα-dependent accumulation of ROS is a strong contributing factor in Glioma cell lines responses to fenofibrate. The ROS scavenger, N-acetyl-cysteine (NAC), restored cell motility, improved mitochondrial potential, and increased ATP levels in fenofibrate treated Glioma cell lines.

Conclusions: Our results indicate that although fenofibrate-mediated inhibition of the IGF-IR may not be sufficient in counteracting Glioma cell dispersal, PPARα-dependent metabolic switch and the resulting ROS accumulation strongly contribute to the inhibition of these devastating brain tumor cells.

BackgroundGlial neoplasms account for nearly 50% of adult primarybrain tumors, and Glioblastoma multiforme (GBM) isconsidered one of the most malignant type of CNStumors [1,2]. GBMs originate from glial cells in the brainand/or spinal cord, and are characterized by rapid cellgrowth, resistance to radio- and chemo-therapies, andrelentless spread of neoplastic cells within the CNS [1].Currently, the treatments that prolong to some extent thesurvival of GBM patients are invasive surgery, andaggressive radiotherapy, followed by chemotherapy(temolozomid [3,4]); treatment with antibodies and

inhibitors (imatinib, getifinib, avastin [5]), or anti-growthfactor therapy (for instance antisense strategies againstIGF-I or TGFβ [6,7]), which increase survival up to 18-24months, instead of 8-11 months of classic survival if onlysurgery and radiotherapy are applied.

GBMs are characterized by a wide variety of genomicabnormalities including loss of heterozygosity for 10q,EGFR amplification and/or mutations, p16 deletions, aswell as p53 and PTEN mutations [8,9]. In addition, theIGF-I receptor (IGF-IR) signaling system has been sus-pected for a quite some time as a contributing factor insupporting malignant growth and invasion of Gliomacells [6,10,11]. It has also been reported that inhibition ofthe IGF-IR expression, either by antisense or triple helixstrategies, triggered apoptotic death in Glioma cells invitro, especially under conditions of anchorage-indepen-

* Correspondence: [email protected] Neurological Cancer Research, Stanley S Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USAFull list of author information is available at the end of the article

© 2010 Drukala et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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dence, and attenuated tumor growth in experimental ani-mals [6,10,11].

Previously, we have demonstrated that activation ofPeroxisome Proliferator Activated Receptor alpha (PPAR-α) by fenofibrate, attenuated signaling responses of theIGF-IR [12]. In addition, fenofibrate inhibited growth andcompromised survival of several Medulloblastoma [12]and Melanoma [13,14] cell lines. In other studies, theanticancer effects of fenofibrate have been demonstratedin colon, breast, endometrial and skin cancer cell lines[15-19]. Fenofibrate is a specific agonist of PPARα, whichbelongs to the family of steroid hormone nuclear recep-tors [20], and is characterized by relatively low systemictoxicity [13,14]. PPAR ligands have been almost exclu-sively associated with the treatment of diabetes, hyperlip-idemia and cardiovascular diseases, as they modulate theexpression of genes regulating glucose and lipid metabo-lism [21]. For instance fenofibrate has been widely used tolower plasma levels of triglycerides and cholesterol, toimprove LDL : HDL ratio, and to prevent atherosclerosis[22]. Although, we still do not fully understand how anti-atherosclerotic effects of fenofibrate could be related toits action against Glioma cell motility, its wide spectrumof lipid modifying actions, including strong activation offatty acid β-oxidation, inhibition of glycolysis [16,23] andROS accumulation [24,25], as well as inhibition of theIGF-IR signal transduction [12], all require further exam-ination.

Since the effects of fenofibrate have not been studied inGliomas, and fenofibrate attenuates IGF-IR signalingpathways, we asked first if fenofibrate action against theIGF-IR could repress malignant dissemination of thesebrain tumor cells. Our present in vitro studies were ini-tially planed to target the IGF-IR signaling pathways, andare not directly related to other aspects of the IGF system,which on the other hand may relate to the immune mech-anism of tumor pathology [6,26]. Here we demonstratethat IGF-I-induced and serum-induced motility ofGlioma cell lines were both severely attenuated by fenofi-brate, which depended, at least partially, on the activationof PPARα. Surprisingly, specific attenuation of the IGF-IRfunction by low molecular weight IGF-IR tyrosine kinaseinhibitor, NVP-AEW541, had only modest effects onGlioma cell motility in serum stimulated LN-229 cells,and had practically no effect on T98G cells. Further anal-yses pointed to the accumulation of the reactive oxygenspecies (ROS) as an additional mechanism of the fenofi-brate action since the ROS scavenger, N-acetyl-cysteine(NAC), effectively restored Glioma cell motility. Ourresults show that in addition to the attenuation of theIGF-IR, fenofibrate action involves accumulation of ROS,loss of mitochondrial membrane potential, and a deficitin ATP production, which taken together may explain thesevere impairment of Glioma cell motility. Further stud-

ies are necessary to determine if indeed treatment withfenofibrate could be effective against Glioma cell disper-sal in the CNS.

MethodsCell CultureThe human Glioma cell lines used in this study include:U-87MG (ATCC# HTB14), U-118MG (ATCC# HTB-15),T98G (ATCC# CRL-1690), LN-18 (ATCC# CRL-2610)and LN-229 (ATCC# CRL-2611). In addition, R600mouse embryo fibroblasts, which express 30,000 of thehuman IGF-IR per cell [27], and primary cultures ofhuman fetal astrocytes (Cambrex) were included as con-trols. Human fetal astrocytes were cultured according tothe manufacturer recommendations (Cambrex). TheGlioma cell lines were maintained as monolayer culturesin DMEM supplemented with 50 U/ml penicillin, 50 ng/ml streptomycin, and 10% fetal bovine serum (FBS) at37°C in a 7% CO2 atmosphere. The cells were made par-tially quiescent by 48 hours incubation in serum-freemedium (SFM) (DMEM supplemented with 0.1% bovineserum albumin). Cell motility and cell signaling weretested by stimulating serum-starved cells with 50 ng/mlof recombinant IGF-I in the presence or absence of 50μM fenofibrate. In some experiments, expression ofPPARα was inhibited by utilizing ON-TARGETplusSMARTpool siRNA against human PPARα: CCCGUUAUCUGAAGAGUUC; GCUUUGGCUUUACGGAAUA;GACUCAAGCUGGUGUAUGA; GGGAAACAUCCA-AGAGAUU (Thermo Scientific).

Western Blot AnalysisTo evaluate phosphorylation levels of the selected IGF-IRsignaling molecules, semi quiescent cultures were stimu-lated with IGF-I and total protein extracts collected. Thefollowing primary anti-phosphospecific antibodies wereutilized: anti-pY612IRS-1 rabbit polyclonal (BioSource,Camarillo, CA); anti-pS473Akt, anti-pT202/Y204Erk1/2,and anti-pS21/9GSK3α/β (Cell Signaling Technology, Inc.Danvers, MA) In addition, anti-PPARα mouse monoclo-nal antibody (Chemicon) was utilized. To monitor load-ing conditions anti-IRS-1 (Upstate USA Inc.,Charlottesville, VA), anti-GSK3β, anti-Akt, anti-Erks(Cell Signaling, Danvers, MA) and anti-Grb-2 antibodieswere used.

Cell Motility AssaysImages of migrating cells were recorded and analyzed bycomputer-aided methods, as previously described[28,29]. Cells were cultured in Corning flasks until theyreached confluency. A cell-free area was introduced byscraping the monolayer with pipette tip. Cell migrationinto cell-free area was evaluated for 10 hours in the pres-ence of 10% FBS (positive control); in serum-free medium


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(SFM, negative control), following IGF-I stimulation (50ng/ml); and in the presence or absence of fenofibrate at aconcentration of 50 μM. In some experiments the cellswere pretreated with N-acetylcysteine (NAC). Tracks ofindividual cells were generated by determining cell dis-placements from time-lapse images taken at 20 minutesintervals during a total observation period of 10 hours.The position of the "cell centroid" was marked by anobserver on a digitized image as previously described[28,30]. Fifty cell tracks were recorded under each of theexperimental conditions tested. The cell trajectories werepresented in circular diagrams with the starting point ofeach trajectory located in the diagram center. The follow-ing parameters characterizing cell locomotion were com-puted for each cell using Mathematical Proceduresincluding: total length of cell trajectory (μm); averagespeed of cell locomotion (μm/min); length of final celldisplacement (μm) i.e. the distance between, first and lastpoint of the cell track; and a ratio of cell displacementlength to cell trajectory length - coefficient of dislocationefficiency (CDE). In addition, cell migration was assessedin Transwell™ Chambers (Corning Corporation, USA)with polycarbonate filters (6.5 mm in diameter; 8.0 μmpore size). The cells were suspended in 200 μl of culturemedium and were treated as indicated in the results sec-tion. After 48 hrs the inserts were washed with PBS, thenon-migratory cells were wiped out with cotton swabs(upper site of the filter), and the filters were fixed andstained with crystal violet : carbol : 25% methanol (1:1:2)mixture for 20 min. The remaining blue-stained cells,which migrated across the membrane were countedunder bright light inverted microscopy.

Luciferase AssayThe transcriptional activity of PPARs in LN-229 humanGlioma cell line was determined by utilizing a JsTkpGL3reporter plasmid, which contains luciferase gene drivenby PPAR responsive element (PPRE), which consists ofthree copies of the J site from apo-AII gene promoter[31]. The activation of PPAR elements was evaluated bydual-Firefly/Renilla luciferase reporter system (Promega,Madison, WI), using Femtomaster FB12 chemiluminom-eter (Zylex. Corp).

Intracellular ROS accumulationViable cells were loaded with 1 mM oxidant sensitive dyeRedox Sensor Red CC-1, and with 50 nM of mitochon-drial specific dye MitoTracker Green FM as previouslydescribed [32]. The images were taken with an invertedNikon Eclipse TE300 microscope equipped with a Retiga1300 camera, motorized Z-axis, Nikon Plan Fluor 40×/1.3 Oil objective, and deconvolution software(SlideBook4). The quantification of intracellular ROS andROS co-localization with mitochondria was calculated

from the entire volume of the cell by utilizing the MaskOperation included in SlideBook4 software, according tomanufacturer instructions (Intelligent Imaging Innova-tions, Denver CO).

Mitochondrial PotentialThis measurement was performed by flow cytometrybased MitoPotential Kit according to the manufacturerprotocol (Guava Easy Cyte). Loss of the mitochondrialinner transmembrane potential (ΔΨm) was evaluated byutilizing the cationic dye JC-1, which gives either green ororange fluorescence depending upon mitochondrialmembrane depolarization [33]. The cells were treatedeither with vehicle (DMSO) or 50 μM fenofibrate. Follow-ing 24 hrs incubation, the cells were harvested bytrypsinization, loaded with JC-1 for 30 min and immedi-ately analyzed by Guava EastCyte flowcytometer usingMito-Potential software (Guava Technologies, Inc).

ATP productionModified methodology described by Gato et al. was fol-lowed [34]. ATP levels were measured by ApoSENSORADP/ATP Ratio Assay Kit according to the manufacturerrecommendations (BioVision). The cells were treatedeither with vehicle (DMSO) or 50 μM fenofibrate. After48 hrs, the cells were harvested by trypsinization and 1 ×104 cell aliquots were resuspended in 100 μl of NucleotideReleasing Buffer, 1 μl of ATP Monitoring Enzyme and 1 μlof ADP Converting Enzyme. The luminometric measure-ment was performed using EnVision multi-plate reader(PerkinElmer).

ResultsDetection of IGF-IR, IRS-1 and PPARα in Glioma cell linesIn view of recent findings, which demonstrated the inhib-itory action of fenofibrate against IGF-IR signaling [12],we evaluated IGF-IR, its major signaling molecule, Insu-lin Receptor Substrate 1 (IRS-1), and PPARα protein lev-els in five human Glioma cell lines in comparison toprimary cultures of human fetal astrocytes. Western blotdepicted in Fig. 1A demonstrates that LN-18 and LN-229Glioma cell lines are characterized by elevated proteinlevels for IGF-IR and IRS-1 in comparison to control fetalastrocytes. In contrast, IGF-IR levels in U87MG, U-118MG and T98G are very low. Interestingly, T98G cellsdespite of very low IGF-IR expression retained elevatedIRS-1. All five human Glioma cell lines demonstrated ele-vated PPARα protein levels in comparison to human fetalastrocytes (Fig. 1B). Importantly, exponentially growingLN-229 cells (in 10% FBS), showed both cytosolic andnuclear PPARα subcellular localization which shiftedtowards the nuclear compartment following 24 hrs cellincubation with fenofibrate (Fig. 1C). Quantitatively, anaverage of 7.6% and 2.8% of the nuclear content (DAPI


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Figure 1 Characterization of IGF-IR, IRS-1 and PPAR-α in human Glioma cell lines. Western blot analysis showing IGF-IR, IRS-1 (Panel A), and PPAR-α protein levels (Panel B) in exponentially growing five human Glioma cell lines in comparison to primary human fetal astrocytes, and R600 mouse embryo fibroblasts, which express 30,000 IGF-IR molecules/cell and high levels of IRS-1. Note that U87MG and U-118MG, which do not express PTEN [8], demonstrate very low levels of the IGF-IR and its major signaling molecule, IRS-1. In T98G, in which activity of PTEN is compromised by point mutation [8], IGF-IR is also very low; however IRS-1 is not affected. Equivalent loading was demonstrated by re-probing membranes with anti-Grb-2 antibody. Panel C: Quantification of PPARα nuclear localization (co-localization with DAPI) in exponentially growing LN-229 cells in the presence (FBS + FF) and absence (FBS) of fenofibrate treatment. The nuclear co-localization was calculated from the entire volume of the nucleus by utilizing Mask Operation included in SlideBook4 software, according to manufacturer instructions (Intelligent Imaging Innovations, Denver Co.). The data represent average number of voxels per nucleus +/- SD, (n = 25). * indicates values significantly different from FBS (p≤0.05). Images below the histogram repre-sent examples of PPARα subcellular distribution. Panel D: PPARα transcriptional activity was evaluated in LN-229 cells by utilizing a dual-Firefly/Renilla luciferase reporter system and Femtomaster FB12 chemiluminometer. Data are presented as mean ± SD calculated from two experiments in triplicates (n = 6). * indicates value statistically significantly different (p≤0.05) from control (FBS; cells treated with vehicle only). Statistical significance between two measurements was determined with the two-tailed Student's t-test.


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labeled) co-localized with PPARα in the presence andabsence of the fenofibrate treatment, respectively (n =25). This 2.7-fold increase in nuclear PPARα was accom-panied by almost 4-fold increase in PPAR-transcriptionalactivity (Fig. 1D), further supporting the possibility ofusing fenofibrate to trigger PPARα-mediated responses inGlioma cells.

Fenofibrate-mediated attenuation of the IGF-IR signaling responsesResults in Fig. 2A demonstrated a strong downregulationof the phosphorylation of IRS-1, Akt, ERKs, and GSK-3βin LN-229 cultured in serum-free medium (SFM). Fol-lowing IGF-I stimulation, all four signaling moleculesbecame highly phosphorylated. We have previouslyreported that fenofibrate inhibited IGF-I-induced phos-phorylation events in Medulloblastoma cell lines [12].Considering that LN-229 Glioma cells responded to IGF-I stimulation, we have examined the effects of fenofibrateon IGF-I-induced phosphorylation events. As shown inFig. 2A, pre-incubation of LN-229 cells with 50 μM feno-fibrate (IGF + FF) attenuated IGF-I-induced phosphory-lation of IRS-1, ERKs, Akt and GSK-3β.

Since IGF-I can also stimulate cell motility [35], weevaluated effects of IGF-I on LN-229 cell motility by uti-lizing cell displacement (Figs. 2Band 2C, upper panels)and wound healing assays (Figs. 2B and 2C, lower panels).The results demonstrate that following scratch inducedmonolayer injury LN-229 migrated into the cell-freeareas with different efficiency (lower panels). The cellscultured in SFM covered only 6.1 +/-2% of the free sur-face, and the cells stimulated with IGF-I covered 57.1 +/-5% of the scratched area over the period of 10 hours. Fol-lowing 24 hrs of cell pre-incubation with 50 μM fenofi-brate, IGF-I-induced cell motility was reduced over 2-fold(from 57.1 +/-5% to 27 +/-6%). Since the percentage ofcells covering the scratched area may result also from cellproliferation, to clarify the contribution of cell motility inthis process, we have included the cell displacementassay. Fig. 2B (upper panel) demonstrates trajectories of50 migrating LN-229 cells in SFM, and following IGF-Istimulation in the presence or absence of fenofibrate. Thecircular diagrams were drawn in the initial point of eachtrajectory placed at the origin of the plot as previouslydescribed [28,29]. The determination of cell trajectoriesis necessary to discriminate between overall cell motility/proliferation (wound healing) and the final effective celldisplacement [30]. The final cell displacement of LN-229migrating in the presence of IGF-I was more than 3-foldgreater than in SFM, and the total length of trajectories incells stimulated by IGF-I increased 1.5-fold in compari-son to SFM (Fig.2B and Table 1). In the presence of feno-fibrate, final cell displacement was reduced by 1.8-foldand the total length of trajectories decreased by 1.4-fold.

This inhibition was associated also with significantdecreases in average speed (from 0.18 μm/min to 0.13μm/min) and CEM (from 0.48 to 0.39) (Tables 1 and 2),further confirming the attenuation of Glioma cell motilityby fenofibrate.

Contribution of IGF-IR inhibition to fenofibrate-mediated action against serum-induced Glioma cell motilitySince fenofibrate inhibited IGF-I-induced phosphoryla-tion events and repressed IGF-I-induced LN-229 cellmotility, we asked whether direct inhibition of the IGF-IRby the specific small molecular weight IGF-IR inhibitor,NVP-AEW541, could have a similar inhibitory action.The results in Fig. 2C show that both IGF-I responsive,LN-229, and non-responsive T98G Gliona cell lines werecharacterized by very active cell migration when culturedin the presence of serum (10% FBS). In particular, 78.5 +/-9% and 94.5 +/-4% of the scratched area were repopu-lated by LN-229 and T98G cells, respectively. In serumstimulated LN-229, fenofibrate treatment decreased cellexpansion into the scratched areas by 15-fold (from 78.5%to 5.1%) and in T98G by 11-fold (from 94.7% to 8.6%).Surprisingly, the IGF-IR inhibitor, NVP-AEW541, wasmuch less effective showing only a modest reduction incell motility: 1.5-fold decrease in LN-229 cells, and 1.1-fold decrease in T98G cells (Fig. 2C lower panel). Corre-sponding trajectories for both cell lines are illustrated ascircular diagrams in Fig. 2C (upper panel). Analyses ofindividual cell trajectories showed that 10% FBS stronglystimulated cell motility of LN-229 and T98G. The averagespeed of movement (i.e. total length of cell trajectory/time), the cell displacement, and the coefficient of move-ment efficiency (CME) are presented in Tables 1 and 2.These results demonstrate that the observed inhibition inthe motile activity of the cells resulted from bothdecrease in speed and polarization of movement. Inhibi-tion of the IGF-IR by 1 μM NVP-AEW541, which hasbeen shown in our previous work to repress IGF-IRtyrosine kinase activity in Medulloblastoma [36], onlypartially attenuated serum-induced LN-229 cell motilityand had practically no effect on T98G cells (Fig. 2C;Tables 1 and 2). However, LN-229 cells showed a 1.5-folddecrease in the average cell displacement after NVP-AEW541 treatment, indicating a partial contribution ofthe IGF-IR in this IGF-I responsive Glioma cell line.

Effects of ROS scavenger NAC on fenofibrate-induced inhibition of cell motility, mitochondrial potential and ATP productionSince attenuation of the IGF-IR signalling responses con-tributed only minimally to the fenofibrate-induced inhi-bition of Glioma cell lines, we asked whether themetabolic action of fenofibrate [16] could explain its anti-invasive potential. This could be relevant, since fenofi-


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Figure 2 Fenofibrate inhibits IGF-induced LN-229 cellular responses. Panels A: Western blot analyses showing phosphorylated (active) forms of IRS-1 (pY612), Akt (pS473), ERKs (pT202/pY204) and GSK-3β (pS21/9) in LN-229 human Glioma cells following 48 hrs serum starvation (SFM); IGF-I stim-ulation (50 ng/ml) for 30 min (IGF), and when IGF-I stimulation was preceded by 24 hrs cell pre-incubation with 50 μM fenofibrate (IGF + FF). Antibodies against non-phosphorylated forms of IRS-1, Akt, ERKs, as well as anti-Grb-2 antibody, were used to demonstrate equal protein loading. Panel B: Upper image: Trajectories of 50 migrating LN-229 cells in SFM, and following IGF-I stimulation in the absence (SFM + IGF) and in the presence (SFM + IGF + FF) of 50 μM fenofibrate. Data are displayed in circular diagrams drawn in the initial point of each trajectory placed at the origin of the plot. The posi-tions of the "cell centroid" from the consecutive frames were used for generation of cell tracks. Quantification of multiple cell motility parameters is given in Table 1. Lower image: Live cell time-lapse imaging of LN-229 cells at 10 hrs after scratching the monolayer culture with the pipette tip. The numbers below phase-contrast images indicate % decrease of the scratched areas (+/- SD, n = 3), calculated from the cell-free area measured at time zero and following 10 hrs of continuous cell migration. Panel C: Upper image: Trajectories of 50 migrating LN-229 and T98G cells the presence of 10% FBS (FBS); 10% FBS + 50 μM fenofibrate (FBS + FF); and 10%FBS + 1 μM IGF-IR inhibitor, NVP-AEW541, (FBS + NVP). Quantification of the cell migration parameters are presented in Table 1. Lower image: Cell migration into the scratched cell-free areas were evaluated at time zero (T0, just after the scratch) and after 10 hrs of continuous cell migration into the cell-free area in the presence of 10%FBS (FBS); 10% FBS + 50 μM fenofibrate (FBS + FF); and 10%FBS + 1 μM IGF-IR inhibitor NVP- AEW541 (NVP). The numbers below phase-contrast images of the scratched monolayer indicate average percentage of the scratched area re-occupied by the migrating cells for 10 hrs (+/-SD, n = 3).


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brate via activation of PPARα is expected to force mito-chondrial fatty acid β-oxidation in tumor cells, which areoften characterized by mitochondrial dysfunction, andstrongly rely on glycolysis as the main energy pathway[16,37,38]. Since dysfunctional mitochondrial respirationand oxidative phosphorylation contribute to ROS accu-mulation [39,40], which may further compromise ATPproduction and repress cell motility, we have evaluatedeffects of fenofibrate on ROS accumulation. As shown in

Fig. 3A, incubation of LN-229 cells with 50 μM fenofi-brate for 24 hours (FF) resulted in a significant accumula-tion of intracellular ROS, demonstrated here by orange/red fluorescence in cells loaded with the redox sensitivefluorescent dye, CC-1 red, and with the mitochondrialmarker, mito-tracker green [41]. Importantly, when ROSscavenger, N-acetyl-cystein (NAC), accompanied thefenofibrate treatment the accumulation of ROS was effec-tively reduced. The intensity of total ROS - associated flu-

Table 1: Analyses of Glioma Individual Cell Displacement

Conditions\Parameters Total length of cell trajectory (μm)

Average speed of cell movement (μm/min)a

Length of final cell displacement (μm)

Average rate of cell displacement (μm/min) b

Coefficient of movement efficiency (CME) c

SFM 76.58 ± 2.05* 0.13 ± 0.003* 17.58 ± 1.50* 0.03 ± 0.002* 0.23 ± 0.02*

SFM + IGF 112.8 ± 3.6** 0.18 ± 0.005** 55.89 ± 3.87** 0.09 ± 0.005** 0.48 ± 0.02**

SFM + IGF + FF 80.5 ± 1.64 0.13 ± 0.002 31.07 ± 1.79** 0.05 ± 0.002** 0.39 ± 0.02**

FBS 105.04 ± 2.9 0.175 ± 0.005 67.30 ± 3.07 0.11 ± 0.005 0.64 ± 0.02

FBS + FF 59.61 ± 1.82* 0.01 ± 0.003* 16.83 ± 1.52* 0.02 ± 0.003* 0.27 ± 0.02*

FBS + NVP 94.47 ± 2.35 0.16 ± 0.005 45.27 ± 2.70* 0.07 ± 0.004* 0.47 ± 0.04*

FBS + FF + NAC 105.51 ± 2.6 0.17 ± 0.005 59.03 ± 2.50 0.09 ± 0.004 0.56 ± 0.02

Individual tracks of LN-229 cell locomotion were generated by determination of cell centroid displacements with time-lapse images taken at 20 minutes intervals during a total observation period of 10 hours. The positions of the cell centroid were marked by an observer on a digitized image. Fifty cell tracks were recorded under each of the experimental conditions tested. The cell trajectories were presented in circular diagrams with the starting point of each trajectory situated in the diagram centre. a: the average speed of cell locomotion is defined as the total length of cell trajectory/time of recording (10 hrs). b: the average rate of cell displacement is defined as the total length of cell displacement from the starting point to the final cell position/time of recording (10 hrs). c: the ratio of cell displacement to cell trajectory length. CME would equal 1 for the cell moving persistently along one straight line in one direction and 0 for a random movement [68]. The values are given as the mean +/-SEM (n = 50). * Statistically different from FBS at p≤0.05. ** Statistically different from SFM at p≤0.05.

Table 2: Analyses of Glioma Individual Cell Displacement.

Conditions\Parameters Total length of cell trajectory (μm)

Average speed of cell movement (μm/min)a

Length of final cell displacement (μm)

Average rate of cell displacement (μm/min) b

Coefficient of movement efficiency (CME) c

FBS 206.3 ± 4.6 0.34 ± 0.07 192.99 ± 5.30 0.32 ± 0.008 0.83 ± 0.04

FBS + FF 83.30 ± 2.6* 0.14 ± 0.05* 58.12 ± 2.10* 0.09 ± 0.003* 0.45 ± 0.02*

FBS + NVP 217.03 ± 6 0.36 ± 0.009 201.31 ± 6.61 0.33 ± 0.01 0.8 ± 0.01

FBS + FF + NAC 132.4 ± 3.4 0.22 ± 0.005 67.7 ± 2.8 0.11 ± 0.005 0.51 ± 0.02

Individual tracks of T98G cell locomotion were generated by determination of cell centroid displacements with time-lapse images taken at 20 minutes intervals during a total observation period of 10 hours. All experimental parameters are identical to those described in the legend to Table 1. The values are given as the mean +/-SEM (n = 50). * Statistically different from FBS at p≤0.05. ** Statistically different from SFM at p≤0.05.


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Figure 3 Effects of fenofibrate on intracellular ROS accumulation . Panel A: Exponentially growing LN229 and T98G cells (in 10%FBS) were treat-ed with 50 μM fenofibrate (FF), in the presence or absence of ROS scavenger, N-acetyl-cysteine (NAC). The cells were loaded with Redox Sensor Red CC-1, and MitoTracker Green FM as previously described [41]. A series of three-dimensional images of each individual picture were deconvoluted to one two-dimensional picture and resolved by adjusting the signal cut-off to near maximal intensity to increase resolution. Note strong increase in cytosolic (red fluorescence) and mitochondria associated (yellow fluorescence) ROS accumulation following fenofibrate treatment (FF), which was effectively prevented by NAC (FF + NAC). Panel B: The quantification of intracellular ROS (voxels per cell) in LN-229 and T98G glioma cell lines, respec-tively. The results were collected from the entire volume of the cell and calculated by utilizing Mask Operation included in SlideBook4 software, ac-cording to manufacturer instructions (Intelligent Imaging Innovations, Denver Co.). The data represent average number of voxels per cell +/- SD, (n = 3). * indicates value significantly different from FBS; ** indicates value significantly different from FF (p ≤ 0.05). Panel C: Cell motility evaluated by cell displacement (upper image) and scratch assay (lower image). Upper image: Trajectories of 50 migrating LN-229 and T98G cells in 10%FBS supplemented with 50 μM fenofibrate in the absence (FBS + FF) and in the presence of NAC (FBS + FF + NAC). Quantification of multiple cell motility parameters is given in Table 1. Lower image: Live cell time-lapse imaging of LN-229 and T98G cells at 10 hrs after scratching the monolayer culture with the pipette tip. The numbers below phase-contrast images indicate % decrease of the scratched areas (+/- SD, n = 3), calculated from the cell-free area measured at time zero and following 10 hrs of continuous cell migration.


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orescence increased 3.2-fold in LN-229 cells (Fig. 3B) and3.7-fold in T98G cells (Fig. 3C) following the treatmentwith fenofibrate (FF). In the presence of NAC (FF +NAC), both IGF-I responsive LN-229, and non-respon-sive T98G cells did not accumulate ROS following fenofi-brate treatment, showing the values for ROS associatedfluorescence even lower than those detected in controlcultures (FBS). These results suggest that fenofibrate-mediated accumulation of ROS happens independentlyfrom the IGF-IR, and that ROS scavenger NAC signifi-cantly counteracted fenofibrate-mediated ROS accumu-lation in both LN-229 and in T98G Glioma cells.

Next, we asked if ROS inhibition by NAC could rescueGlioma cell motility. The results in Fig. 3C and in Tables 1and 2 show that fenofibrate-induced inhibition of LN-229and T98G cell motility was effectively counteracted by 10mM NAC. We have further confirmed NAC-mediatedeffects against fenofibrate by analyzing LN-229 cell inva-siveness in the Transwell™ Chambers. The resultsdepicted in Fig. 4D show that in 10% FBS, LN-229 cellsmigrated across the 8 μm pores very effectively. Thisinvasive propensity was significantly counteracted by thefenofibrate treatment, and was partially neutralized byNAC. Importantly, simultaneous treatment of LN-229and T98G cells with fenofibrate and NAC resulted in par-tial restoration of mitochondrial potential (Fig. 4A and4B), and improved ATP production in fenofibrate treatedcells, confirming the involvement of ROS in fenofibrateinhibitory action/s against Glioma cell lines (Fig. 4C).

Effects of PPARα inhibition on fenofibrate action against Glioma cell motilityTo verify whether fenofibrate-mediated ROS accumula-tion and inhibition of Glioma cell motility depends onPPARα, we have utilized PPARα siRNA. Results in Fig. 5Ashow that 48 hrs cell preincubation with 100 and 200 μMSmartPool siRNA designed to target specifically PPARαmRNA, resulted in almost 5-fold and over 20-folddecrease PPARα protein levels, respectively. Importantly,this strong PPARα inhibition counteracted fenofibrate-induced accumulation of ROS (Fig. 5B), and effectivelyrescued LN-229 cell motility in the presence of fenofi-brate. In summary, our results show a strong inhibitoryaction of fenofibrate against Glioma cell motility. Thisinhibitory action relies on ROS accumulation and ismediated at least partially by the activation of PPARα. Incontrast, downregulation of the IGF-IR induced by feno-fibrate has only a modest contribution to the inhibition ofGlioma cell motility despite the fact that IGF-I stimulatesinvasiveness of IGF-I-responsive LN-229 cells.

DiscusionOur presented work is an in vitro study in which we eval-uate the involvement of IGF-IR and ROS in fenofibrate/

PPARα -mediated inhibition of Glioma cell motility. Ourexperimental setting is based on human glioma cellmodel obtained from ATCC, and although our resultscannot be directly extrapolated the existing mechanismsthat control Glioblastoma invasiveness in vivo, we haveshown their potential usefulness for future clinicalresearch studies.

Here, we have evaluated cellular and molecularresponses of Glioma cells to fenofibrate, and we attemptto discuss its potential use as a new therapeutic agentagainst Glioblastoma. In this respect our preliminarystudies (not shown) demonstrate elevated levels ofPPARα in multiple Glioblastoma clinical samples. Inter-estingly, PPARα was detected preferentially in the cyto-plasm of the tumor cells, and nuclear PPARα was foundonly in restricted areas of the tumor adjacent to thenecrotic tumor tissue. This prominent presence of cyto-solic PPARα, which belongs to the family of nuclear ste-roid receptors, may indicate that its transcriptionalactivity in Glioblastomas is low in comparison to thenuclear PPARα detected in the control normal brain tis-sues in which both neurons and astrocytes were positive(preliminary observations). This may also suggest thatGlioblastoma cells require exogenous stimulation to acti-vate/translocate PPARα to the nucleus. Indeed, theresults in Fig. 1D confirmed enhanced PPARα transcrip-tional activity following fenofibrate treatment, which wasaccompanied by increased detection of PPARα in thenuclei of LN-229 Glioma cells (Fig. 1C).

Since in our previous studies fenofibrate attenuatedIGF-IR in Medulloblastoma cell lines [12], we are askinghere if fenofibrate could compromise this signaling path-way in human Glioma cell lines. We have selected LN-229and T98G human Glioma cell lines, which express highand low levels of the IGF-IR, respectively (Fig. 1A). Incontrast to T98G, LN-229 cells responded to IGF-I stim-ulation by elevated cell proliferation (data not shown),and increased cell motility (Fig. 2B). Since, theseresponses of LN-229 cells were effectively blocked byfenofibrate, we suspected first that fenofibrate-mediatedattenuation of the IGF-IR signaling is responsible for itsinhibitory action. Interestingly, fenofibrate also inhibitedserum-induced cell motility not only in IGF-I sensitiveLN-229 cells, but also in T98G cells, which do notrespond well to IGF-I stimulation. Surprisingly, serum-stimulated LN-229 and T98G cells were both resistant tosmall molecular weight IGF-IR inhibitor, NVP-AEW541,which effectively inhibited growth and survival of severalother tumor cell lines including Medulloblastoma, colonand prostate cancer [36,42,43]. These minimal effects ofIGF-IR inhibition on Glioma cell motility could explainonly moderate clinical results obtained in the treatmentof malignant astrocytomas using antisense strategies [44].Antisense strategies in which immune response rather


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Figure 4 Effects of fenofibrate and NAC on mitochondrial function. Panels A and B: Mitochondrial potential was evaluated in LN-229 and T98G cells, respectively, by utilizing flowcytometry based MitoPotential Kit according to manufacturer's protocol (Guava EasyCyte). Loss of mitochondrial inner transmembrane potential (ΔΨm) was evaluated by a cationic dye JC-1 that gives either green or orange fluorescence depending upon mito-chondrial membrane depolarization. The cells growing in 10% FBS were treated either with vehicle (DMSO) or with 50 μM fenofibrate in the absence (FBS + FF50) or in the presence of NAC (FBS + FF50 + NAC). Following 24 hrs incubation, the cells were loaded with JC-1 for 30 minutes and analyzed by Guava EastCyte flowcytometer using Mito-Potential software. Note that fenofibrate treatment increases percentage of cells with compromised mi-tochondrial potential. Quantification of the mitochondrial potential is shown in the last panel. Data represent average percentage of cells showing polarized or depolarized mitochondria +/- SD, (n = 3). Panel C: ATP levels were evaluated by ApoSENSOR ADP/ATP Ratio Assay Kit (BioVision). The luminometric measurement was performed using EnVision multi-plate reader (PerkinElmer). Data are presented as mean ± SD calculated from two experiments in triplicates (n = 6). * indicates values statistically different from FBS. ** indicates values statistically different from FF, (p ≤ 0.05). Note a strong inhibition of ATP production following 48 hrs cell exposure to 50 μM fenofibrate (FF), which was effectively prevented by the ROS scavenger, NAC. Panels D: Effects of IGF-I, fenofibrate and NAC on LN-229 cell migration evaluated in Transwell Chambers. The cells were seeded at the density of 5 × 104/chamber in 200 μl of 10%FBS containing culture medium (control). The cells were additionally treated with 50 μM fenofibrate either in the absence (FF) or in the presence of NAC (FF + NAC). In addition, we have evaluated cell migration in serum-free medium (SFM) and in SFM supple-mented with IGF-I (50 ng/ml). Data are presented as mean ± SD from three independent experiments in duplicates (n = 6). Statistical significance was tested between control and FF (*), between FF and FF + NAC (**), and between SFM and SFM + IGF (***); p≤0.05.


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Figure 5 Effects of PPARα siRNA on fenofibrate-mediated ROS accumulation and cell motility. Panel A: Western blot analysis showing PPARα protein levels in LN-229 cells incubated with 200 μM of irrelevant siRNA against nuclear lamins (NL: 200 μM), and with 100 and 200 μM of ON-TARGRT plus SMARTpool siRNA against human PPARα (Thermo Scientific). The histogram below indicates densitometric analysis of the blot analyzed by EZQuant-Gel 2.17 (EZQuant Biology Software Solutions, Tel Aviv, Israel). Panel B: ROS accumulation evaluated in exponentially growing LN229 cells (FBS). The cells were treated with 50 μM fenofibrate (FF), in the presence or absence of 200 μM PPARα siRNA (siPPARα). The cells were loaded with Redox Sensor Red CC-1, and MitoTracker Green FM as previously described [41]. The quantification of intracellular ROS (voxels per cell) is illustrated below. The results were collected from the entire volume of the cell and calculated by utilizing Mask Operation included in SlideBook4 software, ac-cording to manufacturer instructions (Intelligent Imaging Innovations, Denver Co.). The data represent average number of voxels per cell +/- SD, (n = 3). * indicates value significantly different from FBS; ** indicates value significantly different from FF (p≤0.05). Panel C: LN-229 cell migration evaluated in Transwell Chambers. Experimental conditions are similar to those described in the legend to Fig. 4D. Exponentially growing LN-229 cells (in 10% FBS) were treated with 50 μM fenofibrate (FF) in the presence or in the absence of 200 μM siRNA against PPARα. After 48 hrs the cells, which did not migrate through the pores, were removed and cells on the bottom surface of the filters were fixed, stained and counted. Data are presented as mean ± SD from three independent experiments in duplicates (n = 3). Statistical significance was tested between FBS (control) and FF (*), and between FF and FF + PPAR siRNA (**); p≤0.05.


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than IGF-IR or TGFβ inhibition per se were suggested aremore effective [6,45]. Further, we speculate that althoughIGF-I contributes to the malignant spread of LN-229cells, NVP-AEW541 was not effective since other growthpromoting mechanism/s, in addition to the IGF-IR, couldbe involved in supporting dissemination of these tumorcells. Despite of this resistance to IGF-IR inhibition, feno-fibrate effectively inhibited Glioma cell motility in thepresence of 10% FBS. Further experiments pointed to theaccumulation of reactive oxygen species (ROS) as a possi-ble mechanism of the fenofibrate action, since the ROSscavenger, NAC, effectively restored LN-229 cell motility,improved mitochondrial potential and enhanced ATPproduction in fenofibrate treatment cultures of LN-229cells.

Another aspect of IGF-IR function is its role in protect-ing tumor cells from apoptosis [46-48]. Indeed, differentstrategies aiming against the IGF-IR were often associ-ated with apoptotic death of different types of tumorcells, including Gliomas [6] and Medulloblastomas[36,49]. Note however, pro-apoptotic effects of IGF-IRinhibition were observed either when tumor cells werecultured in the condition of anchorage-independence[36,50] or when IGF-IR inhibition was used to sensitizetumor cells to other anticancer treatments [51-53]. In ourexperimental setting the treatment of Glioma cells byfenofibrate, which attenuates IGF-IR signaling wasapplied to monolayer cultures, the condition in whichtumor cells are quite resistant to apoptosis. Indeed, wedid not observed any significant increase in Glioma apop-totic cell death even in the presence of 50 μM fenofinrate,the concentration, which effectively inhibited both cellmotility and IGF-I -mediated phosphorylations (Fig. 2).

So far, our results indicate that specific inhibition of theIGF-IR affects only minimally Glioma cell motility (Fig.2C), which makes them very different from Medulloblas-toma cell lines in which inhibition of the IGF-IR was suf-ficient to attenuate their growth and survival in achorage-independence [12,36]. Although the mechanism by whichfenofibrate attenuates IGF-IR is still under investigation,our preliminary observations suggest that fenofibrate uti-lizes a PPARα independent mechanism in repressing thistyrosine kinase receptor. In this regard, fenofibrate hasbeen shown to increase plasma membrane rigidity in amanner similar to elevated cholesterol content in cellmembranes [54]. In this report, fenofibrate did notchange the membrane content of cholesterol, butincreased plasma membrane rigidity, altering activities ofintegral membrane proteins such as the endoplasmicreticulum Ca2+-ATPase and γ-secretase-mediated cleav-age of APP [54]. Further experiments are required todetermine whether similar fenofibrate-mediated changesin the fluidity of plasma membrane are indeed responsi-ble for attenuation of the ligand-induced clustering of the

IGF-IR, a critical step in auto-phosphorylation of thereceptor molecules and the initiation of growth promot-ing signaling cascades.

Despite of our seemingly contradictory findings, i.e.,that IGF-I treatment induces Glioma cell motility, how-ever, the same cells are resistant to the specific IGF-IRinhibitor; and that fenofibrate attenuates IGF-IR signalingresponses, the fenofibrate treatment was still very effec-tive in compromising glioma cell motility. Therefore,alternative mechanism/s of the fenofibrate action shouldbe considered. One possibility is that fenofibrate anti-cancer action could be associated with an aberrant cancercell energy metabolism. This idea originates from the pio-neering work of Otto Warburg who demonstrated a dis-tinctive dependence of tumor cells from glycolysis, evenwhen there is sufficient amount of oxygen available formuch more effective oxidative phosphorylation [38,55].Only recently, it has been established that the inclinationof tumor cells for glycolysis is mainly driven by mito-chondrial dysfunction [56,57]. A direct link betweenmitochondrial aerobic respiration and carcinogenesishave been provided by the demonstration that the loss ofp53 function, which is the most commonly mutated genein cancer [8], including Gliomas, results in the decreaseof synthesis of cytochrome C oxidase expression (SCO2)[58]. SCO2 is crucial for the incorporation of mitochon-drial DNA-encoded cytochrome C oxidase subunit(MTCO2) into the cytochrome C oxidase complex. Theproper assembly of this complex is essential for the mito-chondrial respiration. Therefore, SCO2 deficit in p53-deficient cells heavily impairs oxidative phosphorylationand may trigger the switch towards glycolysis [58].

In respect to the anti-cancer properties of fenofibrate,activated PPARα, which is a transcriptional activator ofthe fatty acid β-oxidation machinery [16], could switchenergy metabolism towards fatty acid degradation, anddecrease glucose uptake by repressing glucose trans-porter GLUT4 [21,59]. Additionally, increased rate of oxi-dation of fatty acids and ketone bodies forces the declinein glucose utilization through the inhibition of glycolyticenzymes [60,61]. This could be highly relevant to theGlioma cells since their energy metabolism and the abilityto migrate is mitochondria independent and stronglyrelies on glycolysis [62]. Therefore, one could speculatethat in glucose-dependent Glioma cells [62] with partialmitochondrial dysfunction, fenofibrate could force anaberrant mitochondrial oxidative phosphorylation lead-ing to ROS accumulation, oxidative damage, and severedeficit in ATP production.

In this respect our results indicate that indeed treat-ment with fenofibrate was associated with ROS accumu-lation (Fig. 3), which could be explained by the aberrantfunction of the mitochondrial electron respiratory chainat the level of NADH cytochrome C reductase [63], or


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elevated xanthine oxidase expression [64], and cytosolicROS, by elevated peroxisomal β-oxidation or microsomalω-oxidation [64,65].

Fenofibrate is also known to be responsible for a strongPPARα-dependent induction of mitochondrial uncou-pling proteins, e.g. UCP2 [66] in various cell models,therefore the decreased mitochondrial membrane poten-tial observed in the fenofibrate treated LN-229 cellsmight be attributed to this event as well. Since the Gliomacell lines used in this study show much higher levels ofPPARα expression than control astrocytes, PPARα drivenUCP2 expression is not unlikely. UCP2 acting as a proto-nophore facilitates passive proton flow through the mito-chondrial inner membrane, which results in uncouplingrespiration from ATP production. Moreover, UCP2 hasbeen shown to act as a metabolic sensor, which promotesthe switch from glucose dependent metabolism towardsfatty acid and glutamine oxidation [67]. These two effectsmay additionally contribute to the Glioma cell energydepletion, which was manifested here by a severe inhibi-tion of cell motility.

Since ROS scavenger, N-acetyl-cysteine (NAC), as wellas siRNA against human PPARα prevented ROS accumu-lation, enhanced ATP production, and restored LN-229cell motility, we have concluded that PPARα inducedmetabolic switch towards mitochondria could be themajor contributing factor in the observed anti-canceraction of fenofibrate. Therefore, in addition to the impair-ment of the IGF-IR signaling responses, Glioma cellstreated with fenofibrate could be brought to the verge ofmetabolic dysfunction by forcing mitochondrial oxidativerespiration in the tumor cells, which strongly depend onglycolysis. This opens an opportunity for the use ofPPARα agonists, including fenofibrate, since it should beselectively toxic for tumor cells and relatively harmlessfor cells with normal mitochondrial function.

ConclusionsOur results show strong inhibition of Glioma cell motilityin vitro by fenofibrate, which involves ROS accumulation,severe mitochondrial dysfunction and a deficit in ATPproduction. The involvement of IGF-IR inhibition in thisprocess was less apparent despite of IGF-I supportingrole in glioma cell motility. Since fenofibrate has relativelylow systemic toxicity, its potential clinical use againstbrain tumors including GBMs should be considered.

Competing interestsThe authors declare that they have no competing interest.

Authors' contributionsJD, ZM and EW carried out cell migration assays and performed detailed quan-tification of cell motility. LDV responsible for immunohistochemistry and analy-ses of the clinical samples; KU, AW and MG carried out IGF-I signaling as well asROS accumulation and ATP production assays. KR wrote the manuscript, was

responsible for design of the experiments and interpretation of the data. Allauthors read and approved final version of the manuscript.

AcknowledgementsGrant support: NIH awarded to KR (RO1CA095518); LDV (P01NS036466 -Neuro-pathology Core); and by grant awarded to JD (N N302 061936) from the Polish Ministry of Science and Higher Education. We would like to thank Drs. Martin White and George Tuszynski for their insightful comments and editorial help, and Jin Ying Wang for technical assistance.

Author Details1Neurological Cancer Research, Stanley S Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA, 2Department of Genetics, University of Pennsylvania, School of Medicine, Philadelphia, PA, USA, 3Department of Cell Biology, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland and 4Department of Food Biotechnology, Faculty of Food Technology, Agricultural University of Krakow, Krakow, Poland

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Received: 8 December 2009 Accepted: 22 June 2010 Published: 22 June 2010This article is available from: http://www.molecular-cancer.com/content/9/1/159© 2010 Drukala et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Molecular Cancer 2010, 9:159

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doi: 10.1186/1476-4598-9-159Cite this article as: Drukala et al., ROS accumulation and IGF-IR inhibition contribute to fenofibrate/PPAR? -mediated inhibition of Glioma cell motility in vitro Molecular Cancer 2010, 9:159











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